Uses of diterpenoid triepoxides as an anti-proliferative agent

ABSTRACT

Combinations of diterpenoid triepoxides and anti-proliferative agents are used in a combination therapy to treat hyperproliferative disorders. Anti-proliferative agents of interest include agents active in killing tumor cells, as well as immunosuppressants, and a variety of other agents that reduce cellular proliferation in targeted tissues. Synergistic combinations provide for comparable or improved therapeutic effects, while lowering adverse side effects.

BACKGROUND

Progress in the treatment of solid tumors has been slow and sporadicdespite the development of new chemotherapeutic agents. There are manyroadblocks to successful chemotherapy, including drug resistance,resistance to apoptosis, and the inactivation of tumor suppressor genes.Some human cancers are drug resistant before treatment begins, while inothers drug resistance develops over successive rounds of chemotherapy.

One type of drug resistance, called multidrug resistance, ischaracterized by cross resistance to functionally and structurallyunrelated drugs. Typical drugs that are affected by the multidrugresistance are doxorubicin, vincristine, vinblastine, colchicine,actinomycin D, and others. At least some multidrug resistance is acomplex phenotype that is linked to a high expression of a cell membranedrug efflux transporter called Mdr1 protein, also known asP-glycoprotein. This membrane “pump” has broad specificity and acts toremove from the cell a wide variety of chemically unrelated toxins.

Another factor in cancer therapy is the susceptibility of targeted cellsto apoptosis. Many cytotoxic drugs that kill cells by crippling cellularmetabolism at high concentration can trigger apoptosis in susceptiblecells at much lower concentration. Increased susceptibility to apoptosiscan be acquired by tumor cells as a byproduct of the genetic changesresponsible for malignant transformation, but most tumors tend toacquire other genetic lesions which abrogate this increased sensitivity.Either at presentation or after therapeutic attempts, the tumor cellscan become less sensitive to apoptosis than vital normal dividing cells.Such tumors are generally not curable by conventional chemotherapeuticapproaches. Although decreased drug uptake, altered intracellular druglocalization, accelerated detoxification and alteration of drug targetare important factors, pleiotropic resistance due to defective apoptoticresponse is also a significant category of drug resistance in cancer.

An important tumor suppressor gene is the gene encoding the cellularprotein, p53, which is a 53 kD nuclear phosphoprotein that controls cellproliferation. Mutations to the p53 gene and allele loss on chromosome17p, where this gene is located, are among the most frequent alterationsidentified in human malignancies. The p53 protein is highly conservedthrough evolution and is expressed in most normal tissues. Wild-type p53has been shown to be involved in control of the cell cycle,transcriptional regulation, DNA replication, and induction of apoptosis.

Various mutant p53 alleles are known in which a single base substitutionresults in the synthesis of proteins that have quite different growthregulatory properties and, ultimately, lead to malignancies. In fact,the p53 gene has been found to be the most frequently mutated gene incommon human cancers, and is particularly associated with those cancerslinked to cigarette smoke. The overexpression of p53 in breast tumorshas also been documented.

An area to search for new therapeutic interventions is that oftraditional Chinese medicines. One of these traditional medicines isfrom Tripteryguim wilfordii Hook F, a shrub-like vine from theCelastraceae family. A variety of preparations derived from this planthave been used in South China for many years to treat different forms ofarthritis and other autoimmune diseases. In 1978, an extract ofTripterygium wilfordii Hook F was produced by chloroform methanolextraction of the woody portion of the roots and designated T2. Reportsin the Chinese literature describe T2 treatment of more than 750patients with a variety of autoimmune diseases.

The Chinese experience has suggested that a daily dosage of about 1mg/kg of T2 is safe and effective as an immunosuppressant. Acute andchronic toxicity studies have been carried out in China using a varietyof animal models. The LD₅₀ in mice was reported to be around 150 mg/kg.The toxicity studies suggest that T2 exhibits a reasonable safety indexand should be able to be administered to patients safely.

The development of chemotherapeutic agents and combinations of agentsthat avoid problems of drug resistance and resistance to apoptosis areof great interest for the treatment of cancer.

Relevant Literature

The isolation, purification, and characterization of immunosuppressivecompounds from tripterygium: triptolide and tripdiolide is reported byGu et al. (1995) Int J Immunopharmacol 17(5):351-6. Yang et al. (1998)Immunopharmacology 40(2):139-49 provide evidence that suggests theimmunosuppressive agent triptolide inhibits antigen or mitogen-induced Tcell proliferation, and induces apoptotic death of T cell hybridomas andperipheral T cells. Shamon et al. (1997) Cancer Lett 112(1):113-7evaluate the antitumor potential of triptolide. Tengchaisri et al.(1998). Cancer Left. 133(2):169-75 evaluate the antitumor activity oftriptolide against cholangiocarcinoma growth in vitro and in hamsters.

Lee et al. (1999) J Biol Chem 274(19):13451-5 describe the interactionof PG490 (triptolide) with tumor necrosis factor-alpha to induceapoptosis in tumor cells. Triptolide was found to inhibit T-cellinterleukin-2 expression at the level of purine-box/nuclear factor ofactivated T-cells and NF-kappa B transcriptional activation by Qiu etal. (1999) J Biol Chem. 274(19):13443-50.

SUMMARY OF THE INVENTION

Compositions and methods are provided for the use of diterpenoidtriepoxides in combination with anti-proliferative agents, as acombination therapy to treat hyperproliferative disorders. The methodsand compositions are particularly useful in the treatment of multi-drugresistant tumor cells. Anti-proliferative agents of interest includeagents active in killing tumor cells, as well as immunosuppressants, anda variety of other agents that reduce cellular proliferation in targetedtissues. The targeted cells are contacted with an anti-proliferativeagent and diterpenoid triepoxides, e.g. triptolide, tripdiolide, etc.,or prodrugs that convert to such compounds under physiologicalconditions, either locally or systemically. Synergistic combinationsprovide for comparable or improved therapeutic effects, while loweringadverse side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the cytotoxicity of PG490 in tumor cells.

FIG. 2 is a graph depicting the inhibition of PG490-induced apoptosis.

FIG. 3 illustrates the effect of PG490-88 on preestablished H23 tumors.

FIG. 4 depicts the effect of PG490-88 on preestablished Dx5 MDR tumors.

FIG. 5. Triptolide inhibits Mdm2 gene expression.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Diterpenoid triepoxides are formulated in combination withanti-proliferative agents, as a combination therapy to treathyperproliferative disorders. Although the diterpenoid triepoxides, andthe anti-proliferative agents, are active when administered alone, theconcentrations required for a killing dose may create unacceptable sideeffects. The methods and compositions are particularly useful in thetreatment of multi-drug resistant tumor cells.

Anti-proliferative agents of interest include agents active in killingtumor cells, as well as immunosuppressants, and a variety of otheragents that reduce cellular proliferation in targeted tissues. Thetargeted cells are contacted with an anti-proliferative agent andditerpenoid triepoxides, e.g. triptolide, tripdiolide, etc., or prodrugsthat convert to such compounds under physiological conditions, eitherlocally or systemically. Synergistic combinations provide for comparableor improved therapeutic effects, while lowering adverse side effects.The subject methods provide a means for therapeutic treatment andinvestigation of hyperproliferative disorders, through the induction ofa novel cell-killing pathway. Animal models, particularly small mammals,e.g. murine, lagomorpha, etc. are of interest for experimentalinvestigations.

The subject methods are used for prophylactic or therapeutic purposes.The term “treatment” as used herein refers to reducing or alleviatingsymptoms in a subject, preventing symptoms from worsening orprogressing, inhibition or elimination of the causative agent, orprevention of the disorder in a subject who is free therefrom. Forexample, treatment of a cancer patient may be reduction of tumor size,elimination of malignant cells, prevention of metastasis, or theprevention of relapse in a patient who has been cured. The treatment ofongoing disease, to stabilize or improve the clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.

In one aspect of the invention, the targeted cell population is a tumorcell population. Particular benefits are obtained when the targetedcells express functional p53 protein. Triptolide is shown to induce p53protein expression in several wild-type p53 tumor cell lines, andwild-type p53 significantly enhanced the cytotoxicity of triptolide.However, functional p53 is not required for triptolide-inducedapoptosis.

In one embodiment of the invention, the anti-proliferative agent is aDNA-damaging agent, such as nucleotide analogs, e.g. purines andpyrimidines, alkylating agents, etc. Another anti-proliferative agent ofparticular interest is taxol.

Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

Hyperproliferative disorders: refers to excess cell proliferation,relative to that occurring with the same type of cell in the generalpopulation and/or the same type of cell obtained from a patient at anearlier time. The term denotes malignant as well as non-malignant cellpopulations. Such disorders have an excess cell proliferation of one ormore subsets of cells, which often appear to differ from the surroundingtissue both morphologically and genotypically. The excess cellproliferation can be determined by reference to the general populationand/or by reference to a particular patient, e.g. at an earlier point inthe patient's life. Hyperproliferative cell disorders can occur indifferent types of animals and in humans, and produce different physicalmanifestations depending upon the affected cells.

Hyperproliferative cell disorders include cancers; blood vesselproliferative disorders such as restenosis, atherosclerosis, in-stentstenosis, vascular graft restenosis, etc.; fibrotic disorders;psoriasis; inflammatory disorders, e.g. arthritis, etc.; glomerularnephritis; endometriosis; macular degenerative disorders; benign growthdisorders such as prostate enlargement and lipomas; and autoimmunedisorders. Cancers are of particular interest, including leukemias,lymphomas (Hodgkins and non-Hodgkins), sarcomas, melanomas, adenomas,carcinomas of solid tissue, hypoxic tumors, squamous cell carcinomas ofthe mouth, throat, larynx, and lung, genitourinary cancers such ascervical and bladder cancer, hematopoietic cancers, head and neckcancers, and nervous system cancers, benign lesions such as papillomas,and the like.

Multidrug resistant cells: Cells of particular interest for the subjectanti-proliferative therapy are multi-drug resistant. Multi-drugresistance is frequently caused by an integral glycoprotein in theplasma membrane of the targeted cell, P-glycoprotein(pleiotropic-glycoprotein, Pgp, MDR1), or a related homolog (MRP). Whenexpressed by tumor cells, MDR1 expels cytotoxic chemotherapeutic agents,and thus allows the tumor cell to survive anticancer treatments even athigh drug doses.

Various methods may be used to determine whether a particular tumor cellsample is multi-drug resistant. Multi-drug resistance can be diagnosedin tumors by molecular biology techniques (gene expression at the mRNAlevel), by immunological techniques (quantification of P-glycoproteinitself) or by functional approaches (measuring dye exclusion). Thesequence of P-glycoprotein may be obtained as Genbank accession numberNM_(—)000927 (Chen et al. (1986) Cell 47:381-389.

In MDR1-expressing cells a decreased uptake of cytotoxic drugs can bevisualized by measuring the cellular accumulation or uptake offluorescent compounds, e.g., anthracyclines (Herweijer et al. (1989)Cytometry 10:463-468), verapamil-derivatives (Lelong et al. (1991) Mol.Pharmacol. 40:490-494), rhodamine 123 (Neyfakh (1988) Exp. Cell Res.174:168-174); and Fluo-3 (Wall etal. (1993) Eur. J. Cancer29:1024-1027). Alternatively, the sample of cells may be exposed to acalcein compound; measuring the amount of calcein compound accumulatingin the specimen cells relative to control cells. Reduced calceinaccumulation in specimen cells relative to control cells indicates thepresence of multi-drug resistance in the biological specimen.

diterpenoid triepoxide sensitizing agent: compounds of interest for usein the combination therapy include compounds having the structure:

wherein X₁ is OH, ═O; or OR¹;

X₂ and X₃ are independently OH, OR¹ or H;

R¹ is —C(O)—Y—Z, wherein Y is a branched or unbranched C₁ to C₆ alkyl oralkenyl group; and Z is COOR², NR³R³, or +NR⁴R^(4′)R^(4″), where R² is acation; R³ and R^(3′) are independently H or branched or unbranched C₁to C₆ alkyl, hydroxyalkyl, or alkoxyalkyl, or R³ and R^(3′) takentogether form a 5- to 7-member heterocyclic ring whose ring atoms areselected from the group consisting of carbon, nitrogen, oxygen andsulfur, wherein the ring atoms include 2 to 6 carbon atoms, or morenitrogen atoms, and optionally one or more oxygen or sulfur atoms, andwherein the ring is unsubstituted or is substituted with one or moregroups selected from R⁵, OR⁵, NR⁵R⁶, SR⁵, NO₂, CN, C(O)R⁵, C(O)NR⁵R⁶,and halogen (fluoro, chloro, bromo, or iodo), where R⁵ and R⁶ areindependently hydrogen, lower alkyl or lower alkenyl; and R⁴, R^(4′) andR^(4″) are independently branched or unbranched C₁ to C₆ alkyl,hydroxyalkyl or alkoxyalkyl. Examples of such molecules may be found inInternational Patent application WO98/52951, and WO97/31921, hereinincorporated by reference.

Compounds of particular interest include triptolide, tripdiolide,triptonide, tripterinin, 16-hydroxytriptolide, triptriolide, andtripchloride; as well as derivatives of triptolide, 16-hydroxytriptolideand tripdiolide (2-hydroxytriptolide) that are derivatized at one ormore hydroxyl groups. Such derivatives may be ester derivatives, wherethe attached ester substituents include one or more amino or carboxylategroups. Prodrugs of particular interest include triptolide succinatesodium salt and triptolide succinate tris(hydroxy-methyl)aminomethanesalt.

The compounds of the invention may be prepared from triptolide,tripdiolide, or 16-hydroxytriptolide obtained from the root xylem of theChinese medicinal plant Tripterygium wilfordii or from other knownsources. Methods for preparing triptolide and related compounds areknown in the art.

Anti-proliferative agents: agents that act to reduce cellularproliferation are known in the art and widely used. Such agents includealkylating agents, such as nitrogen mustards, e.g. mechlorethamine,cyclophosphamide, melphalan (L-sarcolysin), etc.; and nitrosoureas, e.g.carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU),streptozocin, chlorozotocin, etc. Such agents are used in the treatmentof cancer, as well as being immunosuppressants and anti-inflammatoryagents.

Antimetabolite agents include pyrimidines, e.g. cytarabine (CYTOSAR-U),cytosine arabinoside, fluorouracil (5-FU), floxuridine (FUdR), etc.;purines, e.g. thioguanine (6-thioguanine), mercaptopurine (6-MP),pentostatin, fluorouracil (5-FU) etc.; and folic acid analogs, e.g.methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, etc. Methotrexateis widely used as an immunosuppressant, particularly with allogeneicorgan transplants, as well as in the treatment of otherhyperproliferative disorders. Leucovorin is useful as an anti-infectivedrug.

Other natural products include azathioprine; brequinar; alkaloids, e.g.vincristine, vinblastine, vinorelbine, etc.; podophyllotoxins, e.g.etoposide, teniposide, etc.; antibiotics, e.g. anthracycline,daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine),idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.;phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides,e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithrmycin);anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g.mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506(tacrolimus, prograf), rapamycin, etc.; and the like.

Hormone modulators include adrenocorticosteroids, e.g. prednisone,dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesteronecaproate, medroxyprogesterone acetate, megestrol acetate, estradiol,clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g.aminoglutethimide. Estrogens stimulate proliferation anddifferentiation, therefore compounds that bind to the estrogen receptorare used to block this activity. Corticosteroids may inhibit T cellproliferation.

Other chemotherapeutic agents include metal complexes, e.g. cisplatin(cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines,e.g. N-methylhydrazine. Other anti-proliferative agents of interestinclude immunosuppressants, e.g. mycophenolic acid, thalidomide,desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF105685), etc., taxols, e.g. paclitaxel, etc.

Retinoids, e.g. vitamin A, 13-cis-retinoic acid, trans-retinoic acid,isotretinoin, etc.; carotenoids, e.g. beta-carotene, vitamin D, etc.Retinoids regulate epithelial cell differentiation and proliferation,and are used in both treatment and prophylaxis of epithelialhyperproliferative disorders.

Angiotensinase inhibitors diminish exposure of the mesangium to proteinfactors that stimulate mesangial cell proliferation, and are useful withrespect to vascular proliferative disorders.

CPT-11 is useful as a co-therapeutic agent, e.g. in the treatment ofcolon cancer.

Pharmaceutical Formulations: The diterpenoid triepoxides, and theanti-proliferative agents can be incorporated into a variety offormulations for therapeutic administration. The diterpenoid triepoxideand anti-proliferative agent can be delivered simultaneously, or withina short period of time, by the same or by different routes. In oneembodiment of the invention, a co-formulation is used, where the twocomponents are combined in a single suspension. Alternatively, the twomay be separately formulated.

Part of the total dose may be administered by different routes. Suchadministration may use any route that results in systemic absorption, byany one of several known routes, including but not limited toinhalation, i.e. pulmonary aerosol administration; intranasal;sublingually; orally; and by injection, e.g. subcutaneously,intramuscularly, etc.

More particularly, the compounds of the present invention can beformulated into pharmaceutical compositions by combination withappropriate pharmaceutically acceptable carriers or diluents, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration of the compounds can be achieved invarious ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration. The active agent may be systemic after administration ormay be localized by the use of regional administration, intramuraladministration, or use of an implant that acts to retain the active doseat the site of implantation.

In pharmaceutical dosage forms, the compounds may be administered in theform of their pharmaceutically acceptable salts. They may also be usedin appropriate association with other pharmaceutically active compounds.The following methods and excipients are merely exemplary and are in noway limiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant containing the therapeutic agent is placed inproximity to the site of the tumor, so that the local concentration ofactive agent is increased relative to the rest of the body.

The term “unit dosage form”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Dosage: The combined used of diterpenoid triepoxides andanti-proliferative agents has the advantages that the required dosagesfor the individual drugs is lower, and the effect of the different drugscomplementary. Depending on the patient and condition being treated andon the administration route, the diterpenoid triepoxides will generallybe administered in dosages of 0.001 mg to 5 mg/kg body weight per day.The range is broad, since in general the efficacy of a therapeuticeffect for different mammals varies widely with doses typically being20, 30 or even 40 times smaller (per unit body weight) in man than inthe rat. Similarly the mode of administration can have a large effect ondosage. Thus for example oral dosages in the rat may be ten times theinjection dose. The dosage for the anti-proliferative agent will varysubstantially with the compound, in accordance with the nature of theagent. Higher doses may be used for localized routes of delivery.

A typical dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

Susceptible tumors: The host, or patient, may be from any mammalianspecies, e.g. primate sp., particularly humans; rodents, including mice,rats and hamsters; rabbits; equines, bovines, canines, felines; etc.Animal models are of interest for experimental investigations, providinga model for treatment of human disease.

Tumors of interest include carcinomas, e.g. colon, prostate, breast,melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasiveoral cancer, non-small cell lung carcinoma, transitional and squamouscell urinary carcinoma, etc.; neurological malignancies, e.g.neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhoodacute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia,malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-celllymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoidhyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichenplanus, etc.; and the like.

Some cancers of particular interest include non-small cell lungcarcinoma. Non-small cell lung cancer (NSCLC) is made up of threegeneral subtypes of lung cancer. Epidermoid carcinoma (also calledsquamous cell carcinoma) usually starts in one of the larger bronchialtubes and grows relatively slowly. The size of these tumors can rangefrom very small to quite large. Adenocarcinoma starts growing near theoutside surface of the lung and may vary in both size and growth rate.Some slowly growing adenocarcinomas are described as alveolar cellcancer. Large cell carcinoma starts near the surface of the lung, growsrapidly, and the growth is usually fairly large when diagnosed. Otherless common forms of lung cancer are carcinoid, cylindroma,mucoepidermoid, and malignant mesothelioma.

The majority of breast cancers are adenocarcinomas subtypes. Ductalcarcinoma in situ is the most common type of noninvasive breast cancer.In DCIS, the malignant cells have not metastasized through the walls ofthe ducts into the fatty tissue of the breast. Infiltrating (orinvasive) ductal carcinoma (IDC) has metastasized through the wall ofthe duct and invaded the fatty tissue of the breast. Infiltrating (orinvasive) lobular carcinoma (ILC) is similar to IDC, in that it has thepotential metastasize elsewhere in the body. About 10% to 15% ofinvasive breast cancers are invasive lobular carcinomas.

Melanoma is a malignant tumor of melanocytes. Although most melanomasarise in the skin, they also may arise from mucosal surfaces or at othersites to which neural crest cells migrate. Melanoma occurs predominantlyin adults, and more than half of the cases arise in apparently normalareas of the skin. Prognosis is affected by clinical and histologicalfactors and by anatomic location of the lesion. Thickness and/or levelof invasion of the melanoma, mitotic index, tumor infiltratinglymphocytes, and ulceration or bleeding at the primary site affect theprognosis. Clinical staging is based on whether the tumor has spread toregional lymph nodes or distant sites. For disease clinically confinedto the primary site, the greater the thickness and depth of localinvasion of the melanoma, the higher the chance of lymph node metastasesand the worse the prognosis. Melanoma can spread by local extension(through lymphatics) and/or by hematogenous routes to distant sites. Anyorgan may be involved by metastases, but lungs and liver are commonsites.

Methods of Use

A combined therapy of diterpenoid triepoxide compounds andanti-proliferative agents is administered to a host suffering from ahyperproliferative disorder. Administration may be topical, localized orsystemic, depending on the specific disease. The compounds areadministered at a combined effective dosage that over a suitable periodof time substantially reduces the cellular proliferation, whileminimizing any side-effects. Where the targeted cells are tumor cells,the dosage will usually kill at least about 25% of the tumor cellspresent, more usually at least about 50% killing, and may be about 90%or greater of the tumor cells present. It is contemplated that thecomposition will be obtained and used under the guidance of a physicianfor in vivo use.

To provide the synergistic effect of a combined therapy, the diterpenoidtriepoxide active agents can be delivered together or separately, andsimultaneously or at different times within the day. In one embodimentof the invention, the diterpenoid triepoxide compounds are deliveredprior to administration of the anti-proliferative agents.

The susceptibility of a particular tumor cell to killing with thecombined therapy may be determined by in vitro testing, as detailed inthe experimental section. Typically a culture of the tumor cell iscombined with a combination of a anti-proliferative agents and aditerpenoid triepoxide at varying concentrations for a period of timesufficient to allow the active agents to induce cell killing. For invitro testing, cultured cells from a biopsy sample of the tumor may beused. The viable cells left after treatment are then counted.

The dose will vary depending on the specific anti-proliferative agentsutilized, type of cells targeted by the treatment, patient status, etc.,at a dose sufficient to substantially ablate the targeted cellpopulation, while maintaining patient viability. In some cases therapymay be combined with stem cell replacement therapy to reconstitute thepatient hematopoietic function.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the array” includes reference to one or more arrays andequivalents thereof known to those skilled in the art, and so forth. Alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the celllines, constructs, and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXPERIMENTAL EXAMPLE 1 In vivo Antitumor Activity of a Derivative ofTriptolide

Materials and Methods

Cells and Transfections.

H23 (non-small cell lung cancer) and ZR-75 (breast cancer) cell lineswere purchased from ATCC. The Bcl-2 expression vector was provided byFred Hutchinson Cancer Research Center, Seattle, Wash. MES-SA and Dx5cell lines were provided by Branimir Sikic (Stanford University). Cellswere cultured in the appropriate medium with 10% FCS supplemented withL-glutamine, penicillin and streptomycin. To examine the effect of Bcl-2on cell survival, the Bcl-2 expression vector or the vector alone wasco-transfected with a β-galactosidase expression vector (Invitrogen,Carlsbad, Calif.) at a 5:1 ratio using lipofectamine plus (GIBCO BRL,Gaithersburg, Md.) into Dx5 cells. After 36 h cells were stained with5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal). Cell survivalwas calculated as number of total cells-blue cells/total number of cellsin a 90 mm² area from duplicate plates and expressed as the mean±S.D.

Cell death reagents and assays. Cell viability was measured by an MTTassay as recently described (Lee et al. (1999) J. Biol. Chem.274:13451-13455. z-VAD-fluoromethylketone (z-VAD.fmk) was obtained fromAlexis Biochemicals, San Diego, Calif. The effect of z-VAD.fmk on cellviability was analyzed by annexin and propidium iodide staining followedby FACS analysis according to the manufacturers protocol (ClontechLaboratories, Palo Alto, Calif.). The analysis of apoptosis inhistologic sections was done by terminal deoxynucleotidyl transferase(TdT)-mediated d-UTP nick end labeling (TUNEL) of slides from paraffinsections of day 3 tumors harvested from the mice 24 h after the secondof two daily treatments with PG490-88 or saline. TUNEL staining was doneaccording to the manufacturer's protocol (Boehringer Mannheim,Indianapolis, Ind.) and then the histology slides were counterstainedwith methyl green. DNA was isolated from cells for analysis ofinternucleosomal DNA laddering followed by agarose gel electrophoresisand ethidium bromide staining.

Purification of PG490 and PG490-88. PG490 (triptolide) is composed ofwhite to off-white crystals, has a melting point of 226-240° C.,produces a single spot on thin layer chromatography, conforms to astandard triptolide preparation by Proton Nuclear Magnetic Resonance, is97% pure by reverse phase HPLC evaluation usingacetonitrile:water:methanol, and is within 0.4% of the theoreticalresult for elemental analysis (66.51% C, 6.43% H compared to thetheoretical values of 66.65% C, 6.71% H).

PG490-88, 14-succinyl triptolide sodium salt prepared semisyntheticallyfrom PG490, is composed of white amorphous powder, has a melting pointof 232-250° C., produces a single spot on thin layer chromatography,conforms to a standard PG490-88 preparation by Proton Nuclear MagneticResonance, and is 98% pure by reverse phase HPLC evaluation usingacetonitrile:methanol:0.006M sodium phosphate pH=3.2. PG490-88 is aprodrug of PG490, with a half-life in mouse serum of <5 min at roomtemperature. Stock solutions of PG490-88 (1 mg/ml) were prepared bydissolution in 0.9% NaCl and sterilized by microfiltration using 0.2 μmpore size filters (Supor Acrodisc 25, Gelman Sciences, Ann Arbor,Mich.). The PG490-88 stock solutions were diluted in 0.9% NaCl for IPadministration.

Doxorubicin (Gensia Laboratories, Ltd., Irvine, Calif.) purchased as astock solution of 200 mg/ml was prepared for IP administration bydilution in 0.9% NaCl. Taxol was prepared by dissolution in ethanol andaddition of an equal volume of cremophor EL (Sigma, St. Louis, Mo.) toproduce a stock solution of 30 mg/ml, which was diluted in 0.9% NaCl forIP administration.

Nude mouse xenograft model. Female NCr nude mice were purchased fromTaconic, Germantown, N.Y., and were generally 20-24 grams when used.Mice were kept in autoclaved filter-top microisolator cages withautoclaved water and sterile food ad lib. The cages were maintained inan isolator unit providing filtered air (Lab Products, Inc., Maywood,N.J.). Tumor cells were grown and harvested as described above. NCr nudemice were injected intradermally with 5×10⁶ tumor cells. In someexperiments, treatment was initiated on the day of tumor cellimplantation. Otherwise, tumor size was monitored, the mice were groupedtogether to constitute a similar mean tumor size in each group in anexperiment, and treatment was initiated. Mice were treated IP daily for5 days per week.

Results

PG490 (triptolide) induces apoptosis in tumor cells in vitro. PG490alone was found to be cytotoxic on tumor cell lines which include H23cells, a non-small cell lung cancer cell line with mutant p53, Dx5cells, an MDR uterine sarcoma cell line derived from the MES-SA parentcell line and ZR-75 cells, a breast cancer cell line. Dx5 cells are100-fold more resistant to doxorubicin and 1000-fold more resistant totaxol than the MES-SA parent cell line (Chen et al. (1994) Cancer Res.54:4980-4987). PG490 at a dosage of 10 ng/ml decreased cell viability by65-70% of cells in the H23 and Dx5 cell lines and by 24% of cells in theZR-75 cell line. PG490 at 20 ng/ml reduced cell viability by greaterthan 80% in all three cell lines (FIG. 1).

In FIG. 1A, ZR-75 (breast cancer), H23 (non-small cell lung cancer) andDx5 (MDR uterine sarcoma) cell lines were treated with PG490 at dosagesshown and harvested 48 h later for analysis of cell viability by an MTTassay. Data is the mean of three experiments±S.D. In FIG. 1B, DNA wasisolated from untreated or PG490-treated cells 16 h after the additionof PG490 followed by agarose gel electrophoresis and ethidium bromidestaining.

No significant difference in sensitivity to PG490 was observed betweenthe Dx5 cell line and its parent MES-SA cell line. To confirm thatPG490-induced cell death was apoptotic, the presence of PG490 inducedDNA laddering in Dx5 cells was examined, and it was found that PG490induced DNA laddering in Dx5 cells which began at 6 h and was maximal by16 h.

PG490 (triptolide) did not cause growth arrest or significantly affectcell cycle progression in Dx5 and H23 cells. Overexpression of Bcl-2 wasobserved to increase the cell survival in PG490-treated Dx5 cells from15% to 72% (FIG. 2). z-VAD.fmk (100 μM), a tetrapeptide caspaseinhibitor, also increased cell viability in PG490-treated Dx5 cells from15% to 68% (FIG. 2). Bcl-2 or vector control was transiently transfectedinto Dx5 cells followed by the addition of PG490 (20 ng/ml) and stained36 h later with X-gal. % cell survival was calculated as totalcells-blue cells/total cells×100. z-VAD.fmk (100 μM) was added to Dx5cells 1 h prior to the addition of PG490 (20 ng/ml) and cells wereharvested for analysis of cell viability 36 h later by annexin andpropidium iodide staining followed by FACS analysis. Data represents themean of three replicates from two independent experiments±S.D.

PG490-88 prevents human tumor development in nude mice. The resultsreported above show cytotoxicity of PG490 on tumor cells in vitro. Toextend these studies to an in vivo setting using human tumor cellxenografts, PG490-88 was used, a more easily administered, water solubleprodrug of PG490. H23 tumor cells were implanted intradermally in nudemice and the animals were left untreated or were injected IP daily withPG490-88 starting at the time of implantation. Tumors arose in {fraction(5/5)} of the untreated mice but no tumors were observed after 5 or 7weeks of dosing with PG490-88 at doses ranging from 0.25 to 0.75mg/kg/day (Table 1). PG490-88 treatment was stopped after week 5 in 3mice per group and was continued for an additional 2 weeks in 2 mice pergroup. A visible tumor arose during the sixth week in one animal in eachgroup in which PG490-88 dosed at 0.5 mg/kg/day or less was stopped butno more visible tumors appeared in these groups after week 6 (Table 1).No visible tumors developed in any of the mice through the 10 weeks ofobservation in mice which received 0.75 mg/kg/day of PG490-88.

TABLE 1 PG490-88 Treatment of Nude Mice Prevents Formation of HumanTumor Xenografts Number of mice in group with a tumor Week 5 Week 6 Week10 Untreated 5/5 5/5 5/5 PG490-88 (mg/kg/day) 0.25 0/5 1/5 1/5 0.375 0/51/5 1/5 0.5 0/5 1/5 1/5 0.75 0/5 0/5 0/5 Nude mice were implanted withH23 tumor cells (day 0). Mice were left untreated, or were injected IPwith PG490-88 daily from the day of tumor cell implantation for 5consecutive days per week. PG490-88 was administered for 5 weeks. Theuntreated group consisted of 5 mice. Three mice in each of the treatmentgroups received PG490-88 for 5 weeks, and 2 mice in each of these groupswere given PG490-88 for 2 additional weeks (7 weeks total). The tumorappeared only in mice # in which treatment had been stopped after fiveweeks.

PG490-88 inhibits the growth of established tumors of H23 human tumorcells and displays enhanced efficacy in combination therapy with taxolH23 tumor cells were implanted intradermally in nude mice. When thetumors reached approximately 100 mm³, daily IP treatment with PG490-88was initiated. PG490-88 inhibited tumor growth in a dose-dependentmanner (FIG. 3). The data in FIG. 3 represents the measurement of H23tumor volume on day 14 after the initiation of treatment. Nude micebearing xenografts of H23 human tumor cells were treated daily as shown.The data represent the means and the standard errors of the means of thetumor volumes as percent of the day 0 tumor volumes for each animalmeasured day 3, 6, 10 and 14 days after the initiation of treatment.There were 5 mice per group.

By day 14, the 0.25 mg/kg/day dose of PG490-88 reduced tumor volume to21% of the volume of the vehicle control. PG490-88 at 0.75 mg/kg/dayprogressively reduced the mean tumor volume from day 3 through day 14,decreasing the mean tumor size by 61% from the initial value at day 0and a decrease of 97% relative to the day 14 vehicle control (FIG. 3).Taxol decreased tumor growth at the higher dose (10 mg/kg/day) but notthe lower dose (5 mg/kg/day), with a day 14 mean tumor volume 42% of thevehicle control (FIG. 3). PG490-88 at 0.25 mg/kg/day plus 10 mg/kg/dayof taxol decreased tumor size by 93% relative to the day 14 vehiclecontrol volume (FIG. 3). Taxol at 15 mg/kg/day was not used because oftoxicity.

A histologic section of an H23 tumor three days after treatment withPG490-88 showed many cells with abundant eosinophilic cytoplasm,pyknotic nuclei with thinning or loss of nuclear membrane and condensedchromatin compared to a pattern of more homogenous spindle-shaped cellswith an increased nuclear:cytoplasmic ratio in the saline-treatedcontrol. Also, many TUNEL-positive cells were seen in thePG490-88-treated group in comparison to saline-treated animals. At day15 after the initiation of treatment with PG490-88, the H23 tumor wasreplaced by fibrous scar tissue with a central area of calcium phosphateprecipitation but the saline-treated control was unchanged in appearancecompared to the day 3 saline-treated control.

PG490-88 Inhibits the Growth of Established Tumors of an MDR Human TumorCell Line.

MDR is a factor in failing to achieve durable chemotherapeutic efficacyin the clinical setting. Using an MDR tumor cell line Dx5 the efficacyof PG490-88 was tested. Nude mice were implanted intradermally with Dx5tumor cells, and treatment was initiated when the tumors reachedapproximately 100 mm³. The mean tumor volume increased more than 10-foldover the 14 days from the beginning of treatment in the groups of micereceiving saline or doxorubicin alone at 2 mg/kg/day (FIG. 4).

The data in FIG. 4 represents the measurement of Dx5 tumor volume on day14 after the initiation of treatment. Nude mice bearing xenografts ofDx5 MDR human tumor cells were treated daily as shown. The datarepresent the means and the standard errors of the means of the tumorvolumes as percent of the day 0 tumor volumes for each animal measuredday 3, 7, 10 and 14 days after the initiation of treatment. There were 5mice in the groups receiving saline or PG490-88 plus doxorubicin, and 4mice in the groups receiving PG490-88 or doxorubicin alone.

PG490-88 at 0.75 mg/kg/day reduced the mean tumor size by 28% in threeof the four mice compared to the day 0 values. One tumor grew by2.8-fold compared with its day 0 value. By day 14, combination treatmentwith PG490-88 and doxorubicin produced a 34% reduction in tumor volumefrom day 0 and a 94% reduction in mean tumor volume relative to the day14 vehicle control volume, with all of the tumors decreasing in sizecompared to the day 0 values.

The in vivo studies described above used PG490-88, a succinate saltprodrug of triptolide which is rapidly converted to triptolide in theserum. The dosage of triptolide, based on a molar comparison toPG490-88, was 70 pg/mouse/week and it was well tolerated. It wasobserved that PG490-88 at a dosage of 0.75 mg/kg completely preventedH23 tumor formation in all mice and tumors did not emerge in any of themice 5 weeks after dosing with PG490-88 was stopped.

PG490-88 also markedly inhibited the growth of preestablished H23 tumorsand induced apoptotic cell death in the tumor cells. Additionally, thecombination of PG490-88 (0.25 mg/kg) plus taxol (10 mg/kg) was moretumoricidal than either agent alone in preventing tumor formation by H23cells. In preestablished tumors derived from the MDR Dx5 cell line PG490markedly inhibited tumor growth and doxorubicin did not interfere withthe tumoricidal activity of PG490-88. There was no observable toxicityin mice treated with PG490-88 (0.75 mg/kg) as measured by a change inbody weight, altered activity or labored respiration.

There has been progress in the treatment of some solid tumors butsignificant increases in long term survival have been limited by thedevelopment of p53 mutant and multidrug resistant tumors and by thetoxicity of chemotherapy. The above results demonstrate that PG490-88alone is a safe and potent tumoricidal agent in vivo against a p53mutant and an MDR tumor, and that the tumoricidal activity of PG490-88is enhanced by treatment with chemotherapeutic agents such as taxol.

Example 2 Triptolide Induces Apoptosis in Solid Tumor Cells and EnhancesChemotherapy-Induced Apoptosis

p53 plays a role in triptolide-induced apoptosis in tumor cell lines.Also, triptolide enhances apoptosis induced by DNA-damagingchemotherapeutic agents through the p53 pathway. However, thetriptolide-mediated increase in p53 results in repression of mdm2 andp21^(CiP1/Waf1) transcription. In addition, the levels of the Mdm2 andp21 protein in triptolide-treated cells decrease late after the additionof triptolide. Interestingly, triptolide induces translation of p53without initially affecting p53 protein stability. These findingsdemonstrate that triptolide-induced apoptosis and its enhancement ofchemotherapy-induced apoptosis in p53 wild-type cells are mediated, atleast in part, by the induction of p53 translation.

Material and Methods

Reagents.

PG490 (triptolide, MW 360) was obtained from Pharmagenesis (Palo Alto,Calif.). A549 (non-small cell lung cancer) and HT1080 (fibrosarcoma)cell lines were from ATCC. MCF-7 (breast cancer) cell line was obtainedfrom Dr. Ron Weigel (Stanford University). Mouse embryonic fibroblasts(p53 +/+ and p53 −/−) cell lines were provided by Dr. Amato J. Giaccia(Stanford University). Doxorubicin, cycloheximide, and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wereobtained from Sigma Chemicals. The mdm2 promoter-luciferase constructpBP100-GL2 was provided by Dr. Louis Noumovski (Stanford University) andwas made by cloning the Bgl II-Hind III fragment from the pBP100CATvector into the pGL2-Basic Vector (Promega, Madison, Wis.). MCF-7 cellswere transfected using lipofectamine Plus reagent from the LifeTechnologies, Inc. Cells were collected and lysates were preparedaccording to the manufacturer's protocol for luciferase assay (PromegaCorp., Madison, Wis.). Antibodies for p53, p₂₁ ^(WAF1/CIP1), Mdm2,Protein phosphatase-1 (PP-1), and Erk-2 were from Calbiochem, Inc (LaJolla, Calif.) and the rabbit polyclonal Bax antibody was from UpstateBiotechnology (Lake Placid, N.Y.).

Cell Culture and Luciferase Assay.

A549 (non-small cell lung cancer), HT-1080 (fibrosarcoma), and MCF-7(breast cancer) cells were cultured in the appropriate media with 10%FCS supplemented with L-glutamine, penicillin, and streptomycin. p53wild-type (+/+) and null (−/−) Mouse Embryonic Fibroblasts (MEFs)transfected with the E1A/Ras were grown in DMEM containing 15% FCSsupplemented with L-glutamine, penicillin, and streptomycin.Transfections were done on MCF-7 cells using the lipofectamine Plusreagent. At 24 hours after transfection, MCF-7 cells were left untreatedor treated with triptolide (20 ng/ml) or doxorubicin (100 nM) for 4, 8,and 16 hours and cells were collected for luciferase assay. Luciferaseactivity was measured in samples with equal protein concentration with aLuminometer (Analytical Luminescence Laboratory, San Diego, Calif.).

Cell Viability Assay.

Cell viability was measured by an3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayas described above. Untreated or cells treated with triptolide and/ordoxorubicin were harvested at the indicated times followed by theaddition of MTT to the cells in a 96-well plate. Cells were solubilizedwith CH₃Cl acidified with 0.1N HCl. The 96-plate was read at awavelength of 590 nm on an iEMS Labsystems plate reader.

RT-PCR.

RNA was prepared from MCF-7 cells using Rneasy Mini Kit from Qiagen Inc.(Valencia, Calif.). cDNAs were prepared using M-MLV reversetranscriptase (Gibco) with 2 μg of total RNA. {fraction (1/20)} of totalcDNA was used in limited (25 cycles) PCR reactions using Taq polymerase(Gibco). The following primer pairs were used: p53 [SEQ ID NO:1]5′-AGTCAGATCCTAGCGTCGAG-3′ and 5′-[SEQ ID NO:2] TCTTCTTTGGCTGGGGAGAG-3′,mdm2, [SEQ ID NO:3] 5′-GTCAATCAGCAGGMTCATCGG-3′ and [SEQ ID NO:4]5′-CMTCAGGMCATCAAAGCCCTC-3′, p21, [SEQ ID NO:5]5′-AGTGGGGCATCATCAAAAAC-3′ and [SEQ ID NO:6]5′-GACTCCTTGTTCCGCTGCTMTC-3′, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH)- [SEQ ID NO:7] 5′-CCCATCACCATCTTCCAG-3′ and [SEQID NO:8] 5′-ATGACCTTGCCCACAGCC-3′.

Immunoblotting.

At 8 hours after triptolide and/or doxorubicin treatment cells wereharvested at the times indicated and lysed using HNET buffer (50 mMHEPES pH 7.5, 100 mM NaCl, 1 mM EGTA, and 1% Triton X-100) supplementedwith 1 mM DTT, 1 mM PMSF and protease inhibitors cocktail (BoehringerMannheim, Germany). 35 μg of protein was loaded on 10% SDS-PAGE followedby transferring to PVDF membrane. Immunoblotting was performed aspreviously described using a p53 mouse monoclonal antibody from OncogeneResearch Products (Lee et al. (1999) J Biol Chem 274:13451-5).

To measure p53 half-life cycloheximide (30 μg/ml) was added to MCF-7cells 30 min after the addition of triptolide and harvested at the timesshown for immunoblot analysis of p53. Immunoblot analysis using otherantibodies was performed as described above. The band intensity wasmeasured by NIH Image 1.62.

Sub-cellular Fractionation of MCF-7 Cells.

After treatment with triptolide (5 or 20 ng/ml) and/or doxorubicin (100nM) cytosolic and nuclear extracts were prepared as previously described(Lee et al. (1988) Gene Anal Tech 5:22-31) and 50 μg of each extract wasused in SDS/PAGE immunoblot analysis of p53.

Metabolic Labeling of MCF-7Cells.

Cells were grown to 80% confluence followed by pretreatment withtriptolide (20 ng/ml) for 6 h in the appropriate medium. Cells werewashed twice with short-term labeling medium (RPMI with 5% dialyzed FCSsupplemented with L-glutamine, penicillin, and streptomycin). To depleteintracellular pools of methionine short-term labeling medium was addedfor 15 min at 37° C., then replaced by short-term labeling mediumcontaining 0.1 mCi/ml [³⁵S] methionine (Amersham, Inc.). Cells werelabeled for 30 min at 37° C. and washed with ice-cold PBS beforeharvesting for immunoprecipitation. The cells were lysed using RIPAbuffer supplemented with protease inhibitors and immunoprecipitatedusing an agarose-conjugated p53 mAb (Ab-6, Oncogene Research Products)followed by 10% SDS-PAGE. The intensity of labeled p53 protein wasmeasured by NIH Image 1.62.

Results

Triptolide Induces Apoptosis in Solid Tumor Cell Lines and EnhancesChemotherapy-induced Apoptosis.

To determine if tumor cell lines are sensitized to chemotherapeuticagents in the presence of triptolide, a topoisomerase 11 inhibitor,doxorubicin, was used. Doxorubicin (100 nM) alone in A549 and HT1080cells caused only a slight decrease in cell viability, 14.3 and 6.4%respectively, after 48 hours of drug treatment (Table 1). However, inHT-1080 cells, the combination of triptolide at 5 ng/ml (2.8 nM) plusdoxorubicin reduced cell viability by 65%, but triptolide at 5 ng/ml ordoxorubicin (100 nM) alone reduced cell viability only by 10% and 6%respectively. Triptolide at 20 ng/ml (11.2 nM) alone reduced cellviability by 74% in HT1080 cells. Also, in A549 cells, the combinationof triptolide at 20 ng/ml plus doxorubicin (100 nM) decreased cellviability by 67% but triptolide and doxorubicin alone decreasedviability only by 35% and 15% respectively.

Additionally, we observed that triptolide enhances cell death in A549cells induced by carboplatinum, another topoisomerase 11 inhibitor. Wealso examined the effect of triptolide (20 ng /ml) alone on the MCF-7breast cancer cell line which contains wild-type p53. We found thattriptolide, 5ng/ml and 20 ng/ml, decreased cell viability by 36% and 70%respectively in MCF-7 cells (Table 2). We have also found thattriptolide alone induces cell death in greater than 80% of cells inother solid tumor cell lines. Thus, triptolide alone is cytotoxic intumor cells and it cooperates with doxorubicin to enhance cell death intumor cell lines.

TABLE 2 Cell viability assay of human tumor cell lines after triptolidetreatment Percent survival^(a) Treatment MCF-7 A549 HT-1080 Triptolide 5ng/ml 63.9 ± 8.1 91.1 ± 3.8 90.4 ± 6.2 Triptolide 20 ng/ml 30.5 ± 7.664.0 ± 8.2 26.0 ± 5.2 Doxorubicin 100 nM  ND^(b) 85.7 ± 9.6 93.6 ± 4.3Triptolide 5 ng/ml + ND 76.5 ± 9.9 35.8 ± 6.7 Doxorubicin 100 nMTriptolide 20 ng/ml + ND  33.6 ± 11.4 15.5 ± 1.4 Doxorubicin 100 nM^(a)cell viability was measured by MTT assay after 48 h as described inMaterials and Methods. ^(b)Not determined.

Triptolide Increases Expression of p53.

p53 mediates cell death responses to cytotoxic stimuli such as hypoxia,irradiation and DNA damaging chemotherapeutic agents. Since triptolidealone is cytotoxic and it cooperates with doxorubicin, it washypothesized that triptolide-induced apoptosis may be mediated by p53.In both MCF-7 and A549 cells, which retain wild-type p53, triptolideincreased p53 steady state protein levels 24 fold in a dose- andtime-dependent manner. In MCF-7 cells doxorubicin induced a 2 foldincrease in p53, and triptolide induced a greater than 4-fold increasein p53 protein. In A549 cells, the combination of triptolide (20 ng/ml)plus doxorubicin (100 nM) at 24 h showed the greatest increase (greaterthan a 12-fold increase) in p53. Triptolide (5 ng/ml) in combinationwith doxorubicin also markedly increased p53 in HT1080 cells. We nextexamined if the increase in the p53 protein level was due to an increasein the p53 mRNA. The levels of the p53 mRNA did not increase in responseto triptolide but, in fact, p53 mRNA was slightly reduced in MCF-7 cellstreated for 16 h with triptolide (FIG. 5A).

In the experiments shown in FIG. 5, RT-PCR (A) was performed using 2 μgof total RNAs extracted from MCF-7 cells. Cells were treated withtriptolide (20 ng/ml) or doxorubicin (100 nM) and harvested after 8 and16 hours. GADPH was used as a loading control. The plasmid pBP100-GL2which contains a p53-binding site in the mdm2 promoter was transientlytransfected into MCF-7 cells, and cellular lysates were used for theluciferase assay (B). The values are an average of three experiments±S.D. Taken together, these data suggest that the increase in p53 ispost-transcriptional in cells undergoing triptolide-induced cell death.

Functional p53 Enhances Triptolide-induced Cell Death.

The outcome of many chemotherapeutic drugs or radiation therapy dependson the functional status of the tumor suppressor p53 gene. To determineif the presence of functional p53 contributes to triptolide-induced celldeath, we used mouse embryonic fibroblasts (MEFs) cells with thewild-type (+/+) or null (−/−) p53 gene. Triptolide at dosages of 5 ng/mlor 10 ng/ml reduced p53+/+MEF cell viability by 48% and 73% respectivelyand by 15% and 50% in p53 (−/−) cells (Table 3). In MEF cells with thewild-type p53, doxorubicin induced 35% more cell death than thosewithout functional p53. Also, the combination of triptolide plusdoxorubicin reduced cell viability by 88% in p53 (+/+) cells but only by55% in p53 (−/−) cells. Therefore, functional p53 plays a role inmediating triptolide-induced cell death.

Expression of Mdm2 and p21 are Down-regulated in Cells Treated withTriptolide.

One model of p53-mediated apoptosis is that upon cellular stresses (suchas DNA damage), p53 is stabilized and this increases expression of genessuch as mdm2, bax, p21^(CiP1/Waf1), and gadd45. Mdm2 negativelyregulates p53 stability by mediating nuclear export via direct proteinbinding and/or ubiquitin/proteosome degradation. In DNA damage (such asγ-irradiation), phosphorylations of p53 on serines 15 and 392 by DNA-PKor ATM interferes with the ability of Mdm2 to bind to p53 and target p53for degradation. This results in stabilization and activation of p53.

To determine if a similar mechanism exists in triptolide-inducedapoptosis, the levels of several genes that are downstream of p53transactivation were examined. When MCF-7 cells were treated withdoxorubicin 100 nM, there was about a 1.5-2 fold increase in the Mdm2mRNA and protein. This increase in Mdm2 paralleled the increase in p53level which also resulted in increases in bax and p21 mRNA.

In cells treated with triptolide, however, there was a time-dependentdecrease in mdm2 mRNA. To measure the effect of triptolide on mdm2 geneexpression, a luciferase vector was used, which contains a consensusp53-binding site from the mdm2 promoter. Despite the high levels of p53in triptolide-treated MCF-7 cells, transactivation of the reporterconstruct decreased by approximately 30% in the presence of triptolide.However, doxorubicin increased transactivation of the Mdm2 by 15% by 16h. The repression of the p53 dependent genes by triptolide is not ageneral effect, since gadd45 and elongation factor 1-alpha (EF-1α),which are also induced by p53, were not affected. Thus, triptolideinduces p53 but represses expression of some p53 dependent genes.

To determine if the absence of an increase in p53 target genes in cellstreated with triptolide is due to the lack of p53 translocation, p53translocation into the nucleus was examined after triptolide treatment.Compared with the cells treated with doxorubicin, where the majority ofp53 is translocated into the nuclei, the majority of p53 in cellstreated with triptolide (20 ng/ml) was also translocated into nuclei.

There was no significant change in the levels of the Mdm2 protein inMCF-7 cells treated with 5 ng/ml of triptolide for 8 or 24 hours buttriptolide reduced cell viability by only 10% at this dosage. There wasan approximately 1.5-fold increase in Mdm2 in MCF-7 cells treated with20 ng/ml of triptolide at 8 h but by 24 h there was almost a completeloss of Mdm2 protein (FIG. 4). Also, there was a 3-fold decrease in thelevel of p21 protein in triptolide-treated MCF-7 cells but nosignificant change in Bax.

Triptolide Induces Translation of p53.

To determine the mechanism by which triptolide induces p53 we examinedthe effect of triptolide on p53 protein stability and translation. Toexamine the effect on stability we examined levels of p53 in thepresence of cycloheximide (30 μg/ml) in MCF-7 cells, a dose which blockstranslation. When cells were pretreated with triptolide for 0.5 h priorto the addition of cycloheximide, there was a slight increase in p53stability at 30 min but there was no difference from untreated cells at60 min. These data suggested that the increased steady-state level ofthe p53 protein in response to triptolide did not result from anincrease in the half-life of the p53 protein. We then examined iftriptolide induces translation of p53 by in vivo [³⁵S]methioninemetabolic labeling of MCF-7 cells. We found, interestingly, thattriptolide induced a 4.9-fold increase in p53 translation (FIG. 5B).Thus, triptolide-induced p53 accumulation is mediated by an increase inp53 translation.

Triptolide Induces Cell Death in almost 70% of MCF-7 Cells and EnhancesChemotherapy-induced Cell Death in A549 and HT1080 Cells.

To delineate possible mechanism(s) of triptolide-mediated apoptosis, therole of the p53 tumor suppressor gene was studied. Triptolide inducedp53 protein expression in several wild-type p53 tumor cell lines andwild-type p53 significantly enhanced the cytotoxicity of triptolide.Interestingly, triptolide induced cell death in over 80% of cells in amutant p53 lung cancer cell line so that functional p53 is not requiredfor triptolide-induced apoptosis. The data presented here suggests thattriptolide alone and in combination with DNA damaging agents mediates ap53-dependent dependent apoptotic pathway in tumor cells with wild-typep53. It was observed that triptolide increased levels of p53 at apost-transcriptional level. This was mediated by a 5-fold increase inp53.

A late decrease in Mdm2 protein in triptolide-treated cells provide anadditional mechanism for the increase in p53, and a possible mechanismfor how triptolide sustains induction of p53 in the presence ofDNA-damaging agents. Triptolide-mediated repression of downstream p53genes may serve to inhibit expression of survival factors such as MAP4and the IGF1 receptor. Since triptolide shows enhanced cytotoxicity incombination with DNA damaging agents, it may also interfere with DNArepair. Triptolide, however, does not induce DNA strand breaks asrevealed by a comet assay.

The above results demonstrate that triptolide induces p53 and thatfunctional p53 enhances triptolide-induced apoptosis. It is also shownthat triptolide enhances the cytotoxicity of DNA damaging agents. Thecytotoxic activity of triptolide alone and its ability to cooperate withother cytotoxic agents represents a novel method to enhance cytolysis ofsolid tumor cells in vivo.

What is claimed is:
 1. A method for treatment of a solid tumor sensitiveto the combination below, the method comprising: contacting a targetedsolid tumor cell population with a synergistic combination of CPT-11(Irinotecan); and triptolide; in a combined dosage effective tosubstantially reduce the numbers of said targeted solid tumor cellpopulation.
 2. The method of claim 1, wherein said solid tumor is ahuman tumor.
 3. The method of claim 1, wherein said solid tumor is acarcinoma.
 4. The method of claim 1, wherein said solid tumor ismulti-drug resistant.
 5. The method of claim 1, wherein said solid tumorexpresses functional p53 protein.
 6. The method of claim 1, wherein saidtriptolide and said CPT-11 are administered in a co-formulation.
 7. Themethod of claim 1, wherein said triptolide and said CPT-11 areseparately formulated.
 8. A composition for the treatment of a solidtumor sensitive to the combination below, comprising: CPT-11(Irinotecan) and triptolide in a synergistic combination.